A(n) __________ is an invading foreign substance that induces the formation of antibodies.

Our adaptive immune system saves us from certain death by infection. An infant born with a severely defective adaptive immune system will soon die unless extraordinary measures are taken to isolate it from a host of infectious agents, including bacteria, viruses, fungi, and parasites. Indeed, all multicellular organisms need to defend themselves against infection by such potentially harmful invaders, collectively called pathogens. Invertebrates use relatively simple defense strategies that rely chiefly on protective barriers, toxic molecules, and phagocytic cells that ingest and destroy invading microorganisms (microbes) and larger parasites (such as worms). Vertebrates, too, depend on such innate immune responses as a first line of defense (discussed in Chapter 25), but they can also mount much more sophisticated defenses, called adaptive immune responses. The innate responses call the adaptive immune responses into play, and both work together to eliminate the pathogens (Figure 24-1). Unlike innate immune responses, the adaptive responses are highly specific to the particular pathogen that induced them. They can also provide long-lasting protection. A person who recovers from measles, for example, is protected for life against measles by the adaptive immune system, although not against other common viruses, such as those that cause mumps or chickenpox. In this chapter, we focus mainly on adaptive immune responses, and, unless we indicate otherwise, the term immune responses refers to them. We discuss innate immune responses in detail in Chapter 25.

The function of adaptive immune responses is to destroy invading pathogens and any toxic molecules they produce. Because these responses are destructive, it is crucial that they be made only in response to molecules that are foreign to the host and not to the molecules of the host itself. The ability to distinguish what is foreign from what is self in this way is a fundamental feature of the adaptive immune system. Occasionally, the system fails to make this distinction and reacts destructively against the host's own molecules. Such autoimmune diseases can be fatal.

Of course, many foreign molecules that enter the body are harmless, and it would be pointless and potentially dangerous to mount adaptive immune responses against them. Allergic conditions such as hayfever and asthma are examples of deleterious adaptive immune responses against apparently harmless foreign molecules. Such inappropriate responses are normally avoided because the innate immune system calls adaptive immune responses into play only when it recognizes molecules characteristic of invading pathogens called pathogen-associated immunostimulants (discussed in Chapter 25). Moreover, the innate immune system can distinguish between different classes of pathogens and recruit the most effective form of adaptive immune response to eliminate them.

Any substance capable of eliciting an adaptive immune response is referred to as an antigen (antibody generator). Most of what we know about such responses has come from studies in which an experimenter tricks the adaptive immune system of a laboratory animal (usually a mouse) into responding to a harmless foreign molecule, such as a foreign protein. The trick involves injecting the harmless molecule together with immunostimulants (usually microbial in origin) called adjuvants, which activate the innate immune system. This process is called immunization. If administered in this way, almost any macromolecule, as long as it is foreign to the recipient, can induce an adaptive immune response that is specific to the administered macromolecule. Remarkably, the adaptive immune system can distinguish between antigens that are very similar—such as between two proteins that differ in only a single amino acid, or between two optical isomers of the same molecule.

Adaptive immune responses are carried out by white blood cells called lymphocytes. There are two broad classes of such responses—antibody responses and cell-mediated immune responses, and they are carried out by different classes of lymphocytes, called B cells and T cells, respectively. In antibody responses, B cells are activated to secrete antibodies, which are proteins called immunoglobulins. The antibodies circulate in the bloodstream and permeate the other body fluids, where they bind specifically to the foreign antigen that stimulated their production (Figure 24-2). Binding of antibody inactivates viruses and microbial toxins (such as tetanus toxin or diphtheria toxin) by blocking their ability to bind to receptors on host cells. Antibody binding also marks invading pathogens for destruction, mainly by making it easier for phagocytic cells of the innate immune system to ingest them.

In cell-mediated immune responses, the second class of adaptive immune response, activated T cells react directly against a foreign antigen that is presented to them on the surface of a host cell. The T cell, for example, might kill a virus-infected host cell that has viral antigens on its surface, thereby eliminating the infected cell before the virus has had a chance to replicate (see Figure 24-2). In other cases, the T cell produces signal molecules that activate macrophages to destroy the invading microbes that they have phagocytosed.

We begin this chapter by discussing the general properties of lymphocytes. We then consider the functional and structural features of antibodies that enable them to recognize and neutralize extracellular microbes and the toxins they make. Next, we discuss how B cells can produce a virtually unlimited number of different antibody molecules. Finally, we consider the special features of T cells and the cell-mediated immune responses they are responsible for. Remarkably, T cells can detect microbes hiding inside host cells and either kill the infected cells or help other cells to eliminate the microbes.

Vertebrates inevitably die of infection if they are unable to make antibodies. Antibodies defend us against infection by binding to viruses and microbial toxins, thereby inactivating them (see Figure 24-2). The binding of antibodies to invading pathogens also recruits various types of white blood cells and a system of blood proteins, collectively called complement (discussed in Chapter 25). The white blood cells and activated complement components work together to attack the invaders.

Synthesized exclusively by B cells, antibodies are produced in billions of forms, each with a different amino acid sequence and a different antigen-binding site. Collectively called immunoglobulins (abbreviated as Ig), they are among the most abundant protein components in the blood, constituting about 20% of the total protein in plasma by weight. Mammals make five classes of antibodies, each of which mediates a characteristic biological response following antigen binding. In this section, we discuss the structure and function of antibodies and how they interact with antigen.

As predicted by the clonal selection theory, all antibody molecules made by an individual B cell have the same antigen-binding site. The first antibodies made by a newly formed B cell are not secreted. Instead, they are inserted into the plasma membrane, where they serve as receptors for antigen. Each B cell has approximately 105 such receptors in its plasma membrane. As we discuss later, each of these receptors is stably associated with a complex of transmembrane proteins that activate intracellular signaling pathways when antigen binds to the receptor.

Each B cell produces a single species of antibody, each with a unique antigen-binding site. When a naïve or memory B cell is activated by antigen (with the aid of a helper T cell), it proliferates and differentiates into an antibody-secreting effector cell. Such cells make and secrete large amounts of soluble (rather than membrane-bound) antibody, which has the same unique antigen-binding site as the cell-surface antibody that served earlier as the antigen receptor (Figure 24-17). Effector B cells can begin secreting antibody while they are still small lymphocytes, but the end stage of their maturation pathway is a large plasma cell (see Figure 24-7B), which continuously secretes antibodies at the astonishing rate of about 2000 molecules per second. Plasma cells seem to have committed so much of their protein-synthesizing machinery to making antibody that they are incapable of further growth and division. Although many die after several days, some survive in the bone marrow for months or years and continue to secrete antibodies into the blood.

The simplest antibodies are Y-shaped molecules with two identical antigen-binding sites, one at the tip of each arm of the Y (Figure 24-18). Because of their two antigen-binding sites, they are described as bivalent. As long as an antigen has three or more antigenic determinants, bivalent antibody molecules can cross-link it into a large lattice (Figure 24-19). This lattice can be rapidly phagocytosed and degraded by macrophages. The efficiency of antigen binding and cross-linking is greatly increased by a flexible hinge region in most antibodies, which allows the distance between the two antigen-binding sites to vary (Figure 24-20).

The protective effect of antibodies is not due simply to their ability to bind antigen. They engage in a variety of activities that are mediated by the tail of the Y-shaped molecule. As we discuss later, antibodies with the same antigen-binding sites can have any one of several different tail regions. Each type of tail region gives the antibody different functional properties, such as the ability to activate the complement system, to bind to phagocytic cells, or to cross the placenta from mother to fetus.

The basic structural unit of an antibody molecule consists of four polypeptide chains, two identical light (L) chains (each containing about 220 amino acids) and two identical heavy (H) chains (each usually containing about 440 amino acids). The four chains are held together by a combination of noncovalent and covalent (disulfide) bonds. The molecule is composed of two identical halves, each with the same antigen-binding site. Both light and heavy chains usually cooperate to form the antigen-binding surface (Figure 24-21).

In mammals, there are five classes of antibodies, IgA, IgD, IgE, IgG, and IgM, each with its own class of heavy chain—α, δ, ε, γ, and μ, respectively. IgA molecules have α chains, IgG molecules have γ chains, and so on. In addition, there are a number of subclasses of IgG and IgA immunoglobulins; for example, there are four human IgG subclasses (IgG1, IgG2, IgG3, and IgG4), having γ1, γ2, γ3, andγ4 heavy chains, respectively. The various heavy chains give a distinctive conformation to the hinge and tail regions of antibodies, so that each class (and subclass) has characteristic properties of its own.

IgM, which has μ heavy chains, is always the first class of antibody made by a developing B cell, although many B cells eventually switch to making other classes of antibody (discussed below). The immediate precursor of a B cell, called a pre-B cell, initially makes μ chains, which associate with so-called surrogate light chains (substituting for genuine light chains) and insert into the plasma membrane. The complexes of μ chains and surrogate light chains are required for the cell to progress to the next stage of development, where it makes bona fide light chains. The light chains combine with the μ chains, replacing the surrogate light chains, to form four-chain IgM molecules (each with two μ chains and two light chains). These molecules then insert into the plasma membrane, where they function as receptors for antigen. At this point, the cell is called an immature naïve B cell. After leaving the bone marrow, the cell starts to produce cell-surface IgD molecules as well, with the same antigen-binding site as the IgM molecules. It is now called a mature naïve B cell. It is this cell that can respond to foreign antigen in peripheral lymphoid organs (Figure 24-22).

IgM is not only the first class of antibody to appear on the surface of a developing B cell. It is also the major class secreted into the blood in the early stages of a primary antibody response, on first exposure to an antigen. (Unlike IgM, IgD molecules are secreted in only small amounts and seem to function mainly as cell-surface receptors for antigen.) In its secreted form, IgM is a pentamer composed of five four-chain units, giving it a total of 10 antigen-binding sites. Each pentamer contains one copy of another polypeptide chain, called a J (joining) chain. The J chain is produced by IgM-secreting cells and is covalently inserted between two adjacent tail regions (Figure 24-23).

The binding of an antigen to a single secreted pentameric IgM molecule can activate the complement system. As discussed in Chapter 25, when the antigen is on the surface of an invading pathogen, this activation of complement can either mark the pathogen for phagocytosis or kill it directly.

The major class of immunoglobulin in the blood is IgG, which is a four-chain monomer produced in large quantities during secondary immune responses. Besides activating complement, the tail region of an IgG molecule binds to specific receptors on macrophages and neutrophils. Largely by means of such Fc receptors (so-named because antibody tails are called Fc regions), these phagocytic cells bind, ingest, and destroy infecting microorganisms that have become coated with the IgG antibodies produced in response to the infection (Figure 24-24).

IgG molecules are the only antibodies that can pass from mother to fetus via the placenta. Cells of the placenta that are in contact with maternal blood have Fc receptors that bind blood-borne IgG molecules and direct their passage to the fetus. The antibody molecules bound to the receptors are first taken into the placental cells by receptor-mediated endocytosis. They are then transported across the cell in vesicles and released by exocytosis into the fetal blood (a process called transcytosis, discussed in Chapter 13). Because other classes of antibodies do not bind to these particular Fc receptors, they cannot pass across the placenta. IgG is also secreted into the mother's milk and is taken up from the gut of the neonate into the blood, providing protection for the baby against infection.

IgA is the principal class of antibody in secretions, including saliva, tears, milk, and respiratory and intestinal secretions. Whereas IgA is a four-chain monomer in the blood, it is an eight-chain dimer in secretions (Figure 24-25). It is transported through secretory epithelial cells from the extracellular fluid into the secreted fluid by another type of Fc receptor that is unique to secretory epithelia (Figure 24-26). This Fc receptor can also transport IgM into secretions (but less efficiently), which is probably why individuals with a selective IgA deficiency, the most common form of antibody deficiency, are only mildly affected by the defect.

The tail region of IgE molecules, which are four-chain monomers, binds with unusually high affinity (Ka ~ 1010 liters/mole) to yet another class of Fc receptors. These receptors are located on the surface of mast cells in tissues and of basophils in the blood. The IgE molecules bound to them function as passively acquired receptors for antigen. Antigen binding triggers the mast cell or basophil to secrete a variety of cytokines and biologically active amines, especially histamine (Figure 24-27). These molecules cause blood vessels to dilate and become leaky, which in turn helps white blood cells, antibodies, and complement components to enter sites of infection. The same molecules are also largely responsible for the symptoms of such allergic reactions as hay fever, asthma, and hives. In addition, mast cells secrete factors that attract and activate white blood cells called eosinophils. These cells also have Fc receptors that bind IgE molecules and can kill various types of parasites, especially if the parasites are coated with IgE antibodies.

In addition to the five classes of heavy chains found in antibody molecules, higher vertebrates have two types of light chains, κ and λ, which seem to be functionally indistinguishable. Either type of light chain may be associated with any of the heavy chains. An individual antibody molecule, however, always contains identical light chains and identical heavy chains: an IgG molecule, for instance, may have either κ or λ light chains, but not one of each. As a result of this symmetry, an antibody's antigen-binding sites are always identical. Such symmetry is crucial for the cross-linking function of secreted antibodies (see Figure 24-19).

The properties of the various classes of antibodies in humans are summarized in Table 24-1.

The binding of an antigen to antibody, like the binding of a substrate to an enzyme, is reversible. It is mediated by the sum of many relatively weak non-covalent forces, including hydrogen bonds and hydrophobic van der Waals forces, and ionic interactions. These weak forces are effective only when the antigen molecule is close enough to allow some of its atoms to fit into complementary recesses on the surface of the antibody. The complementary regions of a four-chain antibody unit are its two identical antigen-binding sites; the corresponding region on the antigen is an antigenic determinant (Figure 24-28). Most antigenic macromolecules have many different antigenic determinants and are said to be multivalent; if two or more of them are identical (as in a polymer with a repeating structure), the antigen is said to be polyvalent (Figure 24-29).

The reversible binding reaction between an antigen with a single antigenic determinant (denoted Ag) and a single antigen-binding site (denoted Ab) can be expressed as

The equilibrium point depends both on the concentrations of Ab and Ag and on the strength of their interaction. Clearly, a larger fraction of Ab will become associated with Ag as the concentration of Ag increases. The strength of the interaction is generally expressed as the affinity constant ( Ka) (see Figure 3-44), where

(the square brackets indicate the concentration of each component at equilibrium).

The affinity constant, sometimes called the association constant, can be determined by measuring the concentration of free Ag required to fill half of the antigen-binding sites on the antibody. When half the sites are filled, [AgAb] = [Ab] and Ka = 1/[Ag]. Thus, the reciprocal of the antigen concentration that produces half the maximum binding is equal to the affinity constant of the antibody for the antigen. Common values range from as low as 5 × 104 to as high as 1011 liters/mole.

The affinity of an antibody for an antigenic determinant describes the strength of binding of a single copy of the antigenic determinant to a single antigen-binding site, and it is independent of the number of sites. When, however, a polyvalent antigen, carrying multiple copies of the same antigenic determinant, combines with a polyvalent antibody, the binding strength is greatly increased because all of the antigen-antibody bonds must be broken simultaneously before the antigen and antibody can dissociate. As a result, a typical IgG molecule can bind at least 100 times more strongly to a polyvalent antigen if both antigen-binding sites are engaged than if only one site is engaged. The total binding strength of a polyvalent antibody with a polyvalent antigen is referred to as the avidity of the interaction.

If the affinity of the antigen-binding sites in an IgG and an IgM molecule is the same, the IgM molecule (with 10 binding sites) will have a much greater avidity for a multivalent antigen than an IgG molecule (which has two binding sites). This difference in avidity, often 104-fold or more, is important because antibodies produced early in an immune response usually have much lower affinities than those produced later. Because of its high total avidity, IgM—the major Ig class produced early in immune responses—can function effectively even when each of its binding sites has only a low affinity.

So far we have considered the general structure and function of antibodies. Next we look at the details of their structure, as revealed by studies of their amino acid sequence and three-dimensional structure.

Comparison of the amino acid sequences of different antibody molecules reveals a striking feature with important genetic implications. Both light and heavy chains have a variable sequence at their N-terminal ends but a constant sequence at their C-terminal ends. Consequently, when the amino acid sequences of many different κ chains are compared, the C-terminal halves are the same or show only minor differences, whereas the N-terminal halves are all very different. Light chains have a constant region about 110 amino acids long and a variable region of the same size. The variable region of the heavy chains (at their N-terminus) is also about 110 amino acids long, but the heavy-chain constant region is about three or four times longer (330 or 440 amino acids), depending on the class (Figure 24-30).

It is the N-terminal ends of the light and heavy chains that come together to form the antigen-binding site (see Figure 24-21), and the variability of their amino acid sequences provides the structural basis for the diversity of antigen-binding sites. The diversity in the variable regions of both light and heavy chains is for the most part restricted to three small hypervariable regions in each chain; the remaining parts of the variable region, known as framework regions, are relatively constant. Only the 5–10 amino acids in each hypervariable region form the antigen-binding site (Figure 24-31). As a result, the size of the antigenic determinant that an antibody recognizes is generally comparably small. It can consist of fewer than 25 amino acids on the surface of a globular protein, for example.

Both light and heavy chains are made up of repeating segments—each about 110 amino acids long and each containing one intrachain disulfide bond. These repeating segments fold independently to form compact functional units called immunoglobulin (Ig) domains. As shown in Figure 24-32, a light chain consists of one variable (VL) and one constant (CL) domain (equivalent to the variable and constant regions shown in the top half of Figure 24-30). These domains pair with the variable (VH) and first constant (CH1) domain of the heavy chain to form the antigen-binding region. The remaining constant domains of the heavy chains form the Fc region, which determines the other biological properties of the antibody. Most heavy chains have three constant domains (CH1, CH2, and CH3), but those of IgM and IgE antibodies have four.

The similarity in their domains suggests that antibody chains arose during evolution by a series of gene duplications, beginning with a primordial gene coding for a single 110 amino acid domain of unknown function. This hypothesis is supported by the finding that each domain of the constant region of a heavy chain is encoded by a separate coding sequence (exon) (Figure 24-33).

A number of fragments of antibodies, as well as intact antibody molecules, have been studied by x-ray crystallography. From these examples, we can understand the way in which billions of different antigen-binding sites are constructed on a common structural theme.

As illustrated in Figure 24-34, each Ig domain has a very similar three-dimensional structure based on what is called the immunoglobulin fold, which consists of a sandwich of two β sheets held together by a disulfide bond. We shall see later that many other proteins on the surface of lymphocytes and other cells, many of which function as cell-cell adhesion molecules (discussed in Chapter 19), contain similar domains and hence are members of a very large immunoglobulin (Ig) superfamily of proteins.

The variable domains of antibody molecules are unique in that each has its particular set of three hypervariable regions, which are arranged in three hypervariable loops (see Figure 24-34). The hypervariable loops of both the light and heavy variable domains are clustered together to form the antigen-binding site. Because the variable region of an antibody molecule consists of a highly conserved rigid framework, with hypervariable loops attached at one end, an enormous diversity of antigen-binding sites can be generated by changing only the lengths and amino acid sequences of the hypervariable loops. The overall three-dimensional structure necessary for antibody function remains constant.

X-ray analyses of crystals of antibody fragments bound to an antigenic determinant reveal exactly how the hypervariable loops of the light and heavy variable domains cooperate to form an antigen-binding surface in particular cases. The dimensions and shape of each different site vary depending on the conformations of the polypeptide chain in the hypervariable loops, which in turn are determined by the sequences of the amino acid side chains in the loops. The shapes of binding sites vary greatly—from pockets, to grooves, to undulating flatter surfaces, and even to protrusions—depending on the antibody (Figure 24-35). Smaller ligands tend to bind to deeper pockets, whereas larger ones tend to bind to flatter surfaces. In addition, the binding site can alter its shape after antigen binding to better fit the ligand.

Now that we have discussed the structure and functions of antibodies, we are ready to consider the crucial question that puzzled immunologists for many years—what are the genetic mechanisms that enable each of us to make many billions of different antibody molecules?

Antibodies defend vertebrates against infection by inactivating viruses and microbial toxins and by recruiting the complement system and various types of white blood cell to kill the invading pathogens. A typical antibody molecule is Y-shaped, with two identical antigen-binding sites at the tips of the Y and binding sites for complement components and/or various cell-surface receptors on the tail of the Y.

Each B cell clone makes antibody molecules with a unique antigen-binding site. Initially, during B cell development in the bone marrow, the antibody molecules are inserted into the plasma membrane, where they serve as receptors for antigen. In peripheral lymphoid organs, antigen binding to these receptors, together with costimulatory signals provided by helper T cells, activates the B cells to proliferate and differentiate into either memory cells or antibody-secreting effector cells. The effector cells secrete antibodies with the same unique antigen-binding site as the membrane-bound antibodies.

A typical antibody molecule is composed of four polypeptide chains, two identical heavy chains and two identical light chains. Parts of both the heavy and light chains usually combine to form the antigen-binding sites. There are five classes of antibodies (IgA, IgD, IgE, IgG, and IgM), each with a distinctive heavy chain (α, δ, ε, γ, and μ, respectively). The heavy chains also form the tail (Fc region) of the antibody, which determines what other proteins will bind to the antibody and therefore what biological properties the antibody class has. Either type of light chain (κ or λ) can be associated with any class of heavy chain, but the type of light chain does not seem to influence the properties of the antibody, other than its specificity for antigen.

Each light and heavy chain is composed of a number of Ig domains—β sheet structures containing about 110 amino acids. A light chain has one variable (VL) and one constant (CL) domain, while a heavy chain has one variable (VH) and three or four constant (CH) domains. The amino acid sequence variation in the variable domains of both light and heavy chains is mainly confined to several small hypervariable regions, which protrude as loops at one end of the domains to form the antigen-binding site.